During a survey in 2003 in Limon Pampa, Rio Chico, Bolivia, lucerne (Medicago sativa) plants showing typical phytoplasma witches’ broom and little leaf symptoms were collected and evaluated by nested polymerase chain reaction (PCR) using 16S rDNA primers P1/P7 and R16F2n/R16R2. The PCR products (~1250 bp) were purified and directly sequenced, and the sequence was compared with those of other phytoplasmas. The sequence (accession number AY725211) had the highest similarity (98%) with the phytoplasma from ash witches’ broom disease, a member of the 16SrI aster yellows group (Candidatus Phytoplasma asteris). This is thought to be the first report of alfalfa witches’ broom disease in Bolivia, and the first report of an isolate of Candidatus Phytoplasma asteris infecting lucerne.